Methods for producing mammalian pluripotent stem cell-derived endodermal cells

ABSTRACT

The present invention relates to the directed differentiation of mammalian pluripotent stem cells, especially human pluripotent stem (hPS) cells, into endodermal cells. In particular, the present invention relates to the treatment of mammalian pluripotent stem cells, especially hPS cells, with a DNA demethylating agent while undergoing differentiation into endodermal. The inventors have, as disclosed herein, found that exposing differentiating mammalian pluripotent stem cells, especially hPS cells, to a DNA demethylating agent leads to an improved morphology and improved yield of endodermal cells. The treatment with a DNA demethylating agent also leads to a significant down-regulation of expression of the stem cell marker Oct4 and to an improved expression of endoderm specific markers, notably sox17, cxcr4 and hhex.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.14/648,336, filed on May 29, 2015, which is the U.S. National Phasefiling of PCT International Application No. PCT/EP2013/0075018, filed onNov. 28, 2013, which claims priority to Denmark application no.PA201270739, filed on Nov. 29, 2012 and U.S. Provisional PatentApplication No. 61/731,241, filed on Nov. 29, 212. The contents of theforegoing applications are incorporated herein by reference in theirentireties.

TECHNICAL FIELD

The present invention relates to the directed differentiation ofmammalian pluripotent stem cells, especially human pluripotent stem(hPS) cells, into cells of the definitive endoderm. In particular, thepresent invention relates to the treatment of mammalian pluripotent stemcells, especially hPS cells, with a DNA demethylating agent whileundergoing differentiation into endoderm. The present inventors have, asdisclosed herein, found that exposing differentiating mammalianpluripotent stem cells, especially hPS cells, to a DNA demethylatingagent leads to an improved morphology and improved yield of endodermalcells. The treatment with a DNA demethylating agent also leads to asignificant down-regulation of expression of the stem cell marker Oct4and to an improved expression of endoderm specific markers, notablysox17, cxcr4 and hhex.

BACKGROUND OF THE INVENTION

Human pluripotent stem cells are expected to revolutionize theaccessibility to a variety of human cell types since they have thecapacity, under appropriate conditions, to self-renew as well as theability to form any type of specialized cells of the three germ layers(endoderm, mesoderm, and ectoderm). Of major interest is the endodermallayer since it gives rise to the intestine, pancreas, liver and lung,i.e. organs of the human body failure or damage of which are associatedwith a great number of disease states and clinical disorders seen today.A great promise thus lies in the in vitro development of organ specifictissue for replacement therapy.

Due to its unique capability among the three germ layers to develop intothe above mentioned organs, the endoderm, more specifically thedefinitive endoderm, plays a central role in the production of organspecific tissue. Thus, there is a constant need to improve thecharacteristics of in vitro derived endodermal cells which eventuallyhas an impact on the quality and quantity of the organ specific cellsand tissue. However, early endoderm development is not well understood,with only a few factors so far identified to drive the differentiationof human pluripotent stem cells towards endoderm. Accordingly, findingfurther, yet unidentified, factors having an influence on endodermaldevelopment is important and will help to optimize the cultivationcondition for in vitro production of cells and tissue of endodermalorgans.

The importance of DNA methylation during normal embryogenesis anddevelopment has long been suspected and the application of DNAdemethylating agents can cause reactivation of large swathes of genes ina genome. Previous work has shown that DNA demethylation can be used todirect differentiation of hES cells towards cardiac fate (Yoon et al2006), presumably by activating genes required for cardiomyogenesiswhich would normally be methylated and silenced.

SUMMARY OF THE INVENTION

The present invention describes improved methods by which mammalianpluripotent stem cells, especially human pluripotent stem (hPS) cells,are differentiated into cells of the definitive endoderm, which possessimproved characteristics compared to endodermal cells obtained bycurrently available state of the art methods.

The present invention provides in a first aspect a method for producingmammalian pluripotent stem cell-derived cells of the definitive endoderm(DE cells), especially human pluripotent stem (hPS) cell-derived cellsof the definitive endoderm (DE cells), wherein mammalian pluripotentstem cells, especially human pluripotent stem cells, are exposed to aDNA demethylating agent.

Thus, a method for producing mammalian pluripotent stem cell-derived DEcells is provided which comprises:

Culturing mammalian pluripotent stem cells under differentiationconditions to obtain DE cells, and

Exposing the differentiating mammalian pluripotent stem cells to a DNAdemethylating agent.

Especially, a method for producing human pluripotent stem (hPS)cell-derived DE cells is provided which comprises:

Culturing human pluripotent stem cells under differentiation conditionsto obtain DE cells, and

Exposing the differentiating human pluripotent stem cells to a DNAdemethylating agent.

During the differentiation of mammalian pluripotent stem cells,especially hPS cells, into definitive endodermal cells, thedifferentiating mammalian pluripotent stem cells, especially hPS cells,are exposed to a DNA demethylating agent, such as 5-aza-2-deoxycytidineor 5-azacytidine, to demethylate sections of the genome and allowtranscriptional activation of genes.

The exposure to said DNA demethylating agent may take place at any timeduring the differentiation of the mammalian pluripotent stem cells,especially hPS cells, into DE cells.

As a result of the methods according to the present invention, cells ofthe definitive endoderm and cell compositions comprising the same areobtained having improved characteristics. Thus, in further aspects, theinvention relates to a definitive endodermal cell(s) obtained by themethod of the invention and to a cell composition(s) comprising, orconsisting of, said endodermal cell(s),

In another aspect, the present invention relates to the further use ofthe definitive endodermal cell(s) or cell composition(s) of theinvention for producing cells or tissue of the intestine, pancreas,liver and/or lung. The definitive endodermal cell(s) or cellcomposition(s) of the invention, may, for instance, be used to producehepatic progenitor cells or fully matured hepatocyte-like cells,including hepatic tissue. The definitive endodermal cell(s) or cellcomposition(s) of the invention, may also be used to produce pancreaticprecursor cells or fully matured pancreatic cells, such as pancreaticstellate cells or Langerhans cells, or pancreatic tissue.

In other aspects, the invention provides the further uses of thedefinitive endodermal cell(s) or cell composition(s) of the invention inpharmaceutical and toxicological screening, such as drug discoveryprocesses or toxicity testing.

In a further aspect, the present invention provides the use of a DNAdemethylating agent in the production of cells of the definitiveendoderm from mammalian pluripotent stem cells, especially humanpluripotent stem cells.

In yet a further aspect, the invention relates to kits useful incarrying out the methods of the invention. Included in this aspect arekits which comprise at least one DNA demethylating agent. It isunderstood that the details given herein with respect to the componentsemployed in the methods of the invention also apply to the componentscomprised by the kits of the invention.

In yet a further aspect, the invention relates to compositions. Suchcompositions are particularly useful for producing cells of thedefinitive endoderm from mammalian pluripotent stem cells, especiallyhuman pluripotent stem. Included in this aspect are compositions whichcomprise at least one DNA demethylating agent and activin, such asactivin A or B. It is understood that the details given herein withrespect to the components employed in the methods of the invention alsoapply to the components comprised by the compositions of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for differentiating mammalianpluripotent stem cells, especially human pluripotent stem cells, intocells of the definitive endoderm, the method comprising culturing saidmammalian pluripotent stem cells, especially hPS cells, in a supportiveculture and differentiation medium containing a DNA demethylating agent.The mammalian stem cells, especially hPS cells, while undergoingdifferentiation into endoderm are thus exposed to a DNA demethylatingagent.

Accordingly, the present invention provides a method for producing cellsof the definitive endoderm from mammalian pluripotent stem cells,especially human pluripotent cells, characterized in that the mammalianpluripotent stem cells, especially hPS cells, while undergoingdifferentiation into endoderm are exposed to a DNA demethylating agent.

The method for producing mammalian pluripotent stem cell-derived cellsof the definitive endoderm, especially hPS cell-derived cells of thedefinitive endoderm, may be described as comprising:

Culturing mammalian pluripotent stem cells, especially human pluripotentstem cells, under differentiation conditions to obtain cells of thedefinitive endoderm, and

Exposing the differentiating mammalian pluripotent stem cells,especially human pluripotent stem cells, to a DNA demethylating agent.

The DNA demethylating agent employed in the method according to theinvention may be any compound that interferes with DNA methyltransferaseenzyme activity. Suitable DNA demethylating agents are ones of thenucleoside-analog type, such as cytidine analogues, e.g.5-aza-2-deoxycytidine (decitabine), 5-azacytidine (azacitidine) orzebularine, and of the non-nucleoside type, such as procaine, RG108,S-5-adenosyl-L-homocysteine, Caffeic acid, Chlorogenic acid,Epogallocatechin gallate, Hydralazine hydrochloride, Procainamidehydrochloride or Psammaplin A.

Thus, the DNA demethylating agent employed in the methods of theinvention may be one of the nucleoside-analogue type. Alternatively, theDNA demethylating agent employed in the methods of the invention may beone of the non-nucleoside type. The DNA demethylating agent employed inthe method of the invention may also be a mixture of both types.

Non-limiting examples of DNA demethylating agents of thenucleoside-analog type which may be employed in the method of thepresent invention are 5-aza-2-deoxycytidine (decitabine), 5-azacytidine(azacitidine) and zebularine. Non-limiting examples of thenon-nucleoside type are procaine, RG108, S-5-adenosyl-L-homocysteine,Caffeic acid, Chlorogenic acid, Epogallocatechin gallate, Hydralazinehydrochloride, Procainamide hydrochloride or Psammaplin A.

Accordingly, the DNA demethylating agent employed in the method of theinvention may be one selected from the group consisting of:5-aza-2-deoxycytidine (decitabine) 5-azacytidine (azacitidine),zebularine, procaine, RG108, S-5-adenosyl-L-homocysteine, Caffeic acid,Chlorogenic acid, Epogallocatechin gallate, Hydralazine hydrochloride,Procainamide hydrochloride, Psammaplin A, and combinations thereof.

The DNA demethylating agent employed in the method of the invention maybe a cytidine analogue, such as e.g. 5-aza-2-deoxycytidine (decitabine),5-azacytidine (azacitidine), zebularine, Pseudoisocytidine,5-fluoro-2-deoxycytidine, 5,6-dihydro-5-azacytidine,2′-deoxy-5,6-dihydro-5-azacytidine, 6-azacytidine,2′,2′-Difluoro-deoxycytidine (gemcitabine), orCytosine-beta-D-arabinofurasonide.

The DNA demethylating agent employed in the method of the invention maythus be a cytidine analogue selected from the group consisting of5-aza-2-deoxycytidine (decitabine), 5-azacytidine (azacitidine),5-fluoro-2-deoxycytidine, 5,6-dihydro-5-azacytidine,2′-deoxy-5,6-dihydro-5-azacytidine, 6-azacytidine and2′,2′-Difluoro-deoxycytidine (gemcitabine),

The DNA demethylating agent employed in the method of the invention maythus be a cytidine analogue selected from the group consisting of5-aza-2-deoxycytidine (decitabine), and 5-azacytidine (azacitidine),Alternatively, the DNA demethylating agent employed in the methods ofthe invention may be a cytidine analogue which is not5-aza-2-deoxycytidine (decitabine) or 5-azacytidine (azacitidine). TheDNA demethylating agent employed in the method of the invention may thusbe a cytidine analogue selected from the group consisting of5-aza-2-deoxycytidine (decitabine), 5-azacytidine (azacitidine),5-fluoro-2-deoxycytidine, 5,6-dihydro-5-azacytidine,2′-deoxy-5,6-dihydro-5-azacytidine, 6-azacytidine and2′,2′-Difluoro-deoxycytidine (gemcitabine).

The DNA demethylating agent employed in the method of the invention maythus be 5-aza-2-deoxycytidine. The DNA demethylating agent may also be5-azacytidine. The DNA demethylating agent may also be zebularine. TheDNA demethylating agent may also be Pseudoisocytidine. The DNAdemethylating agent may also be 5-fluoro-2-deoxycytidine. The DNAdemethylating agent may also be 5,6-dihydro-5-azacytidine. The DNAdemethylating agent may also be 2′-deoxy-5,6-dihydro-5-azacytidine. TheDNA demethylating agent may also be 6-azacytidine. The DNA demethylatingagent may also be 2′,2′-Difluoro-deoxycytidine (gemcitabine). The DNAdemethylating agent may also be Cytosine-beta-D-arabinofurasonide. TheDNA demethylating agent may also be procaine. The DNA demethylatingagent may also be RG108. The DNA demethylating agent may also beS-5-adenosyl-L-homocysteine. The DNA demethylating agent may also beCaffeic acid. The DNA demethylating agent may also be Chlorogenic acid.The DNA demethylating agent may also be Epogallocatechin gallate. TheDNA demethylating agent may also be Hydralazine hydrochloride. The DNAdemethylating agent may also be Procainamide hydrochloride. The DNAdemethylating agent may also be Psammaplin A.

The differentiating hPS cells may not only be exposed to one DNAdemethylating agent, but may also be exposure to one or more further DNAdemethylating agents, such as to a combination of two, three, four,five, six or seven of those mentioned above.

The differentiating mammalian pluripotent stem cells, especially hPScells, may, for instance, be exposed to two of the DNA demethylatingagents mentioned above. The differentiating mammalian pluripotent stemcells, especially hPS cells, may thus be exposed to a combination of5-azacytidine (azacitidine) and 5-aza-2-deoxycytidine (decitabine) or toa combination of 5-azacytidine and zebularine. The differentiatingmammalian pluripotent stem cells, especially hPS cells, may thus beexposed to a combination of 5-azacytidine (azacitidine) and5,6-dihydro-5-azacytidine. The differentiating mammalian pluripotentstem cells, especially hPS cells, may thus be exposed to a combinationof 5-aza-2-deoxycytidine (decitabine) and 5,6-dihydro-5-azacytidine. Thedifferentiating mammalian pluripotent stem cells, especially hPS cells,may thus be exposed to a combination of 5-azacytidine (azacitidine) and2′-deoxy-5,6-dihydro-5-azacytidine. The differentiating mammalianpluripotent stem cells, especially hPS cells, may thus be exposed to acombination of 5-aza-2-deoxycytidine (decitabine) and2′-deoxy-5,6-dihydro-5-azacytidine. The differentiating mammalianpluripotent stem cells, especially hPS cells, may thus be exposed to acombination of 5-azacytidine (azacitidine) and 6-azacytidine. Thedifferentiating mammalian pluripotent stem cells, especially hPS cells,may thus be exposed to a combination of 5-aza-2-deoxycytidine(decitabine) and 6-azacytidine. The differentiating mammalianpluripotent stem cells, especially hPS cells, may thus be exposed to acombination of 5-azacytidine (azacitidine) and2′,2′-Difluoro-deoxycytidine (gemcitabine). The differentiatingmammalian pluripotent stem cells, especially hPS cells, may thus beexposed to a combination of 5-aza-2-deoxycytidine (decitabine) and2′,2′-Difluoro-deoxycytidine (gemcitabine).

The differentiating mammalian pluripotent stem cells, especially hPScells, may also be exposed to a combination of a DNA demethylating agentof the nucleoside-analog type and a DNA demethylating agent of thenon-nucleoside type, such as a combination of 5-aza-2-deoxycytidine andone of procaine, RG108, S-5-adenosyl-L-homocysteine, Caffeic acid,Chlorogenic acid, Epogallocatechin gallate, Hydralazine hydrochloride,Procainamide hydrochloride and Psammaplin A. The differentiatingmammalian pluripotent stem cells, especially hPS cells, may also beexposed to three of the DNA demethylating agents mentioned above. Thedifferentiating mammalian pluripotent stem cells, especially hPS cellsmay thus be exposed to a combination of 5-azacytidine (azacitidine),5-aza-2-deoxycytidine (decitabine) and zebularine.

The differentiating mammalian pluripotent stem cells, especially hPScells, may generally be exposed to the DNA demethylating agent at aconcentration in the range of about 1 nM to about 10 μM, such as in therange of about 1 nM to about 5 μM

The differentiating mammalian pluripotent stem cells, especially hPScells, may, for instance, be exposed to the DNA demethylating agent at aconcentration in the range of about 1 nM to about 1 μM. Thedifferentiating mammalian pluripotent stem cells, especially hPS cells,may also be exposed to the DNA demethylating agent at a concentration inthe range of about 1 nM to about 500 nM. The differentiating mammalianpluripotent stem cells, especially hPS cells, may also be exposed to theDNA demethylating agent at a concentration in the range of about 1 nM toabout 250 nM. The differentiating mammalian pluripotent stem cells,especially hPS cells, may also be exposed to the DNA demethylating agentat a concentration in the range of about 1 nM to about 100 nM. Thedifferentiating mammalian pluripotent stem cells, especially hPS cells,may thus be exposed to the DNA demethylating agent at a concentration inthe range of about 1 nM to about 50 nM. The differentiating mammalianpluripotent stem cells, especially hPS cells, may thus be exposed to theDNA demethylating agent at a concentration in the range of about 1 nM toabout 25 nM. The differentiating mammalian pluripotent stem cells,especially hPS cells, may thus be exposed to the DNA demethylating agentat a concentration in the range of about 1 nM to about 15 nM. Thedifferentiating mammalian pluripotent stem cells, especially hPS cells,may also be exposed to the DNA demethylating agent at a concentration inthe range of about 1 nM to about 10 nM. The differentiating mammalianpluripotent stem cells, especially hPS cells, may also be exposed to theDNA demethylating agent at a concentration in the range of about 5 nM toabout 500 nM. The differentiating mammalian pluripotent stem cells,especially hPS cells, may thus be exposed to the DNA demethylating agentat a concentration in the range of about 5 nM to about 250 nM. Thedifferentiating mammalian pluripotent stem cells, especially hPS cells,may also be exposed to the DNA demethylating agent at a concentration inthe range of about 5 nM to about 100 nM. The differentiating mammalianpluripotent stem cells, especially hPS cells, may also be exposed to theDNA demethylating agent at a concentration in the range of about 5 nM toabout 50 nM. The differentiating mammalian pluripotent stem cells,especially hPS cells, may also be exposed to the DNA demethylating agentat a concentration in the range of about 5 nM to about 25 nM. Thedifferentiating mammalian pluripotent stem cells, especially hPS cells,may also be exposed to the DNA demethylating agent at a concentration inthe range of about 5 nM to about 15 nM, such as in the range of about 10nM. The differentiating mammalian pluripotent stem cells, especially hPScells, may also be exposed to the DNA demethylating agent at aconcentration in the range of about 7.5 nM to about 250 nM. Thedifferentiating mammalian pluripotent stem cells, especially hPS cells,may also be exposed to the DNA demethylating agent at a concentration inthe range of about 7.5 nM to about 100 nM. The differentiating mammalianpluripotent stem cells, especially hPS cells, may also be exposed to theDNA demethylating agent at a concentration in the range of about 7.5 nMto about 50 nM. The differentiating mammalian pluripotent stem cells,especially hPS cells, may also be exposed to the DNA demethylating agentat a concentration in the range of about 7.5 nM to about 25 nM. Thedifferentiating mammalian pluripotent stem cells, especially hPS cells,may also be exposed to the DNA demethylating agent at a concentration inthe range of about 7.5 nM to about 12.5 nM.

In case that, for instance, 5-aza-2-deoxycytidine is employed as the DNAdemethylating agent, the differentiating mammalian pluripotent stemcells, especially hPS cells, may be exposed to it at a concentration inthe range of 1 nM to about 1 μM. The differentiating mammalianpluripotent stem cells, especially hPS cells, may thus be exposed to5-aza-2-deoxycytidine at a concentration in the range of about 1 nM toabout 500 nM. The differentiating mammalian pluripotent stem cells,especially hPS cells, may also be exposed to 5-aza-2-deoxycytidine at aconcentration in the range of about 1 nM to about 250 nM. Thedifferentiating mammalian pluripotent stem cells, especially hPS cells,may also be exposed to 5-aza-2-deoxycytidine at a concentration in therange of about 1 nM to about 100 nM. The differentiating mammalianpluripotent stem cells, especially hPS cells, may thus be exposed to5-aza-2-deoxycytidine at a concentration in the range of about 1 nM toabout 50 nM. The differentiating mammalian pluripotent stem cells,especially hPS cells, may thus be exposed to 5-aza-2-deoxycytidine at aconcentration in the range of about 1 nM to about 25 nM. Thedifferentiating mammalian pluripotent stem cells, especially hPS cells,may thus be exposed to 5-aza-2-deoxycytidine at a concentration in therange of about 1 nM to about 15 nM. The differentiating mammalianpluripotent stem cells, especially hPS cells, may also be exposed to5-aza-2-deoxycytidine at a concentration in the range of about 1 nM toabout 10 nM. The differentiating mammalian pluripotent stem cells,especially hPS cells, may also be exposed to 5-aza-2-deoxycytidine at aconcentration in the range of about 5 nM to about 500 nM. Thedifferentiating mammalian pluripotent stem cells, especially hPS cells,may thus be exposed to 5-aza-2-deoxycytidine at a concentration in therange of about 5 nM to about 250 nM. The differentiating mammalianpluripotent stem cells, especially hPS cells, may also be exposed to5-aza-2-deoxycytidine at a concentration in the range of about 5 nM toabout 100 nM. The differentiating mammalian pluripotent stem cells,especially hPS cells, may also be exposed to 5-aza-2-deoxycytidine at aconcentration in the range of about 5 nM to about 50 nM. Thedifferentiating mammalian pluripotent stem cells, especially hPS cells,may also be exposed to 5-aza-2-deoxycytidine at a concentration in therange of about 5 nM to about 25 nM. The differentiating mammalianpluripotent stem cells, especially hPS cells, may also be exposed to5-aza-2-deoxycytidine at a concentration in the range of about 5 nM toabout 15 nM, such as in the range of about 10 nM. The differentiatingmammalian pluripotent stem cells, especially hPS cells, may also beexposed to 5-aza-2-deoxycytidine at a concentration in the range ofabout 7.5 nM to about 250 nM. The differentiating mammalian pluripotentstem cells, especially hPS cells, may also be exposed to5-aza-2-deoxycytidine at a concentration in the range of about 7.5 nM toabout 100 nM. The differentiating mammalian pluripotent stem cells,especially hPS cells, may also be exposed to 5-aza-2-deoxycytidine at aconcentration in the range of about 7.5 nM to about 50 nM. Thedifferentiating mammalian pluripotent stem cells, especially hPS cells,may also be exposed to 5-aza-2-deoxycytidine at a concentration in therange of about 7.5 nM to about 25 nM. The differentiating mammalianpluripotent stem cells, especially hPS cells, may also be exposed to5-aza-2-deoxycytidine at a concentration in the range of about 7.5 nM toabout 12.5 nM.

Similar concentrations may be used in case that 5-azacytidine orzebularine are employed as the DNA demethylating agent. Similarconcentrations may also be used in case of other cytidine analogues,such as, e.g. Pseudoisocytidine, 5-fluoro-2-deoxycytidine,5,6-dihydro-5-azacytidine, 2′-deoxy-5,6-dihydro-5-azacytidine,6-azacytidine, 2′,2′-Difluoro-deoxycytidine (gemcitabine), orCytosine-beta-D-arabinofurasonide, in particular5-fluoro-2-deoxycytidine, 5,6-dihydro-5-azacytidine,2′-deoxy-5,6-dihydro-5-azacytidine, 6-azacytidine or2′,2′-Difluoro-deoxycytidine (gemcitabine).

The differentiating mammalian pluripotent stem cells, especially hPScells, may be exposed to (or treated with) said agent at any stagebetween pluripotent stem cell stage and endodermal stage. Thus, theexposure to said DNA demethylating agent may take place during thedifferentiation of the mammalian pluripotent stem cells, especially hPScells, into DE cells.

The differentiating mammalian pluripotent stem cells, especially hPScells, are usually exposed to the DNA demethylating agent of thenucleoside-analog type (e.g. 5aza-2deoxycytidine, 5-azacytidine,zebularine) when they show greatest proliferative capacity as evidencedby cell doubling time, such as between days 2 and 7 of differentiation.Thus, the DNA demethylating agent may be added to the differentiationmedium on day 2 of differentiation. The DNA demethylating agent may alsobe added to the differentiation medium on day 3 of differentiation. TheDNA demethylating agent may also be added to the differentiation mediumon day 4 of differentiation. The DNA demethylating agent may also beadded to the differentiation medium on day 5 of differentiation. The DNAdemethylating agent may also be added to the differentiation medium onday 6 of differentiation. DNA demethylation agents of the non-nucleosidetype (e.g. procaine, RG108, S-5-adenosyl-L-homocysteine) can be added atany time in the differentiation protocol since they do not require cellproliferation to have an effect.

The treatment of differentiating mammalian pluripotent stem cells,especially hPS cells, with a DNA demethylating agent has surprisinglybeen found to lead to an improved morphology and improved yield ofdefinitive endodermal cells. Moreover, treatment with a demethylatingagent provides for more pure and homogenous endodermal cellpopulations(FIG. 1A-B). Moreover, treatment with a DNA demethylatingagent also surprisingly leads to a significant down-regulation ofexpression of the stem cell marker Oct4 in endodermal cells (FIGS. 1Cand D) and to an improved protein and gene expression of the DE specificmarkers sox17, cxcr4 and hhex (FIG. 1D). It is believed to be the firsttime that such effects are shown for DNA demethylation and theapplication of growth factors involved in the differentiation ofmammalian pluripotent stem cells, especially hPS cells, towardsdefinitive endoderm. Without to be bound by theory, it is believed thatthe action of the involved growth factors at a genomic level is enhancedby the widespread absence of methylation.

The starting material in the present invention may be any type ofmammalian pluripotent stem cells, such as mammalian embryonic stem cellsor mammalian induced pluripotent stem cells.

The mammalian pluripotent stem cells employed in the present inventionmay, for instance, be human pluripotent stem cells, primate pluripotentstem cells, mouse pluripotent stem cells, rat pluripotent stem cells,canine pluripotent stem cells, feline pluripotent stem cells, procinepluripotent stem cells, bovine pluripotent stem cells or equinepluripotent stem cells.

Especially, the starting material in the present invention may be anytype of human pluripotent stem cells, such as human embryonic stem (hES)cells or human induced pluripotent stem (hiPS) cells.

Accordingly, the human pluripotent stem cells which are used as startingmaterial to obtain DE cells may be human embryonic stem cells. Varioustechniques for obtaining such hES cells are known to the skilled person.Preferably, however, the hES cells for use according to the inventionare ones which have been derived (or obtained) without destruction ofthe human embryo, such as by employing the single blastomere removaltechnique described in e.g. Chung et al (2008), further described byMercader et al. in Essential Stem Cell Methods (First Edition, 2009).Suitable hES cell lines for use are, for example, the cell lines SA167,SA181, SA461 (Cellartis AB, Göteborg, Sweden) which are listed in theNIH stem cell registry, the UK Stem Cell bank and the European hESCregistry and are available on request. Other suitable cell lines for useare those established by Klimanskaya et al. (2006), such as cell linesMA01 and MA09, and Chung et al. (2008), such as cell lines MA126, MA127,MA128 and MA129, which all are listed with the International Stem CellRegistry (assigned to Advanced Cell Technology, Inc. Worcester, Mass.,USA).

Alternatively, the human pluripotent stem cells which may be used asstarting material to obtain the endodermal and/or hepatic progenitorcells may be human induced pluripotent stem cells. Various techniquesfor obtaining such hiPS cells have been described in the scientificliterature, and are thus known to the skilled person [see, e.g.,Takahashi et al. (2007); Zhou et al. (2009); Yu and Thomson inEssentials of Stem Cell Biology (2^(nd) Edition].

The starting material in the present invention may be any type ofmammalian pluripotent stem cells, such as mammalian embryonic stem cellsor mammalian induced pluripotent stem cells, which mammalian pluripotentstem cells are not human pluripotent stem cells.

Suitable conditions for differentiating mammalian pluripotent stemcells, especially hPS cells, into DE cells are known (see, e.g., Hay2008, Brolen 2010 and Duan 2010). WO 2009/013254 A1, for example,describes suitable protocols to obtain cells of the definitive endodermfrom hPS cells (Embodiments 1 to 4).

Generally, in order to obtain endodermal cells, mammalian pluripotentstem cells, especially hPS cells, are cultured in a differentiationmedium comprising activin, such as activin A or B. The differentiationmedium may further include a histone deacetylase (HDAC) inhibitor, suchas Sodium Butyrate (NaB), Phenylbutyrate (PB), valproate, trichostatinA, Entinostat or Panobinstat. The differentiation medium may furthercomprise one or more growth factors, such as FGF1, FGF2 and FGF4, and/orserum, such as FBS or FCS. The differentiation medium may comprise aGSK3-inhibitor, such as, e.g., CHIR99021, or an activator of Wntsignalling, such as Wnt3A. The differentiation medium may furthercomprise a PI3K (Phosphoinositide 3-kinase) inhibitor, such as LY294002.

The concentration of activin is usually in the range of about 50 toabout 150 ng/ml, such as about 80 to about 120 ng/ml. Activin may, forexample, be present in the differentiation medium at a concentration ofabout 50 ng/ml or about 100 ng/ml. The concentration of the HDACinhibitor is usually in the range of about 0.5 to about 2 mM. The HDACinhibitor may, for example, be present in the differentiation medium ata concentration of about 0.5 mM or about 1 mM. The concentration of theone or more growth factors may vary depending on the particular compoundused. The concentration of FGF2, for example, is usually in the range ofabout 2 to about 50 ng/ml, such as about 2 to about 10 ng/ml. FGF2 may,for example, be present in the differentiation medium at a concentrationof about 4 or about 5 ng/ml. The concentration of FGF1, for example, isusually in the range of about 50 to about 200 ng/ml, such as about 80 toabout 120 ng/ml. FGF1 may, for example, be present in thedifferentiation medium at a concentration of about 100 ng/ml. Theconcentration of FGF4, for example, is usually in the range of about 20to about 40 ng/ml. FGF4 may, for example, be present in thedifferentiation medium at a concentration of about 30 ng/ml. Theconcentration of serum, if present, is usually in the range of about 0.1to about 2% v/v, such as about 0.1 to about 0.5%, about 0.2 to about1.5% v/v, about 0.2 to about 1% v/v, about 0.5 to 1% v/v or about 0.5 toabout 1.5% v/v. Serum may, for example, be present in thedifferentiation medium at a concentration of about 0.2% v/v, about 0.5%v/v or about 1% v/v. The concentration of the GSK3 inhibitor, ifpresent, is usually in the range of about 0.1 to about 10 μM, such asabout 0.05 to about 5 μM. The concentration of the activator of Wntsignalling, if present, is usually in the range of about 0.05 to about10 ng/ml, such as about 0.5 to about 5 μM. The concentration of the PI3Kinhibitor, for example, is usually in the range of about 0.1 to 10 μM,such as about 1 to 5 μM.

The differentiation medium may further comprise other supplements suchas PEST and/or GlutaMAX. The differentiation medium may also furthercomprise a ROCK inhibitor. The concentration of PEST is usually in therange of about 0.1 to about 0.5% v/v, such as about 0.1 to about 0.25%v/v. The concentration of GlutaMAX is usually in the range of about 0.5to about 1.5% v/v, such as about 0.75 to 1.25% v/v, e.g. about 1% v/v.The differentiation medium may also further comprise a ROCK inhibitor.The concentration of the ROCK inhibitor is usually in the range of about1 to about 10 μM, such as about 2.5 to about 7.5 μM, e.g., about 5 μM.

The culture medium forming the basis for the differentiation medium maybe any culture medium suitable for culturing mammalian pluripotent stemcells, especially hPS cells, such as RPMI 1640 or advanced RPMI 1640medium, Dulbecco's Modified Eagle Medium (DMEM), HCM medium, HBM mediumor Williams E based medium. Thus, the differentiation medium may be RPMI1640 or advanced RPMI 1640 medium comprising or supplemented with theabove-mentioned components. Alternatively, the differentiation mediummay be DMEM comprising or supplemented with the above-mentionedcomponents. The differentiation medium may thus also be HCM mediumcomprising or supplemented with the above-mentioned components. Thedifferentiation medium may thus also be HBM medium comprising orsupplemented with the above-mentioned components. The differentiationmedium may thus also be Williams E based medium comprising orsupplemented with the above-mentioned components.

For endodermal differentiation, mammalian pluripotent stem cells,especially hPS cells, are normally cultured for up to 10 days in anactivin containing differentiation medium as described above. Themammalian pluripotent stem cells, especially hPS cells, may, forexample, be cultured in said differentiation medium for about 4 to about10 days, such as for about 4 to about 9 days, for about 4 to about 7days or for about 7 to about 9 days.

Basic, non-limiting culture conditions for obtaining cells of thedefinitive endoderm from mammalian pluripotent stem cells, especiallyhPS cells, are provided in Example 2 herein.

Further, the endodermal cells of the present invention may be obtainedunder xeno-free conditions. As such, the starting material employed inthe method of the invention may thus be xeno-free, such as xeno-free hPScells or cell lines which have been obtained or established underanimal-free conditions. Moreover, throughout the method of the inventioncells may be cultured completely under xeno-free conditions, giving riseto truly xeno-free endodermal cells. Such cells or cell line could bedistinguished from a non-xeno free composition by the presence innon-xeno free cells of the non-human sialic acid Neu5Gc or othernon-human markers (Martin et al 2005).

As a result of the methods of the present invention, endodermal cells,e.g., DE cells, are obtained with improved features compared tocurrently available state of the art methods.

The populations of definitive endodermal cells or cell compositionsobtained in accordance to the invention show an improved lowerexpression of stem cell markers like Oct4, compared to cell populationsor cell compositions obtained without treatment with a DNA demethylatingagent. Further, the populations of definitive endodermal cells or cellcompositions show an increased gene expression of a number of markerscharacteristic for definitive endodermal cells, notably sox17, cxcr4 andhhex. Moreover, the obtained populations of definitive endodermal cellsor cell compositions are more pure and homogenous compared to onesobtained without treatment with a DNA demethylating agent.

The cell composition(s) of the invention may further be characterized inthat at least 70% such as e.g. 75%, 80%, 90% or 95% of the cells areendodermal cells of the present invention.

Once obtained, the endodermal cell(s) or cell composition(s) of theinvention may further be used to produce cells or tissue of theintestine, pancreas, liver and/or lung. The endodermal cell(s) or cellcomposition(s) of the invention, may, for instance, be used to producehepatic progenitor cells or fully matured hepatocyte-like cells,including hepatic tissue. The endodermal cell(s) or cell composition(s)of the invention may also be used to produce pancreatic precursor cellsor fully matured pancreatic cells, such as pancreatic stellate cells orLangerhans cells, or pancreatic tissue.

The endodermal cell(s) or cell composition(s) of the invention may alsobe further used in pharmaceutical and toxicological screening, such asdrug discovery processes or toxicity testing.

The invention also provides kits. Such kits are particularly useful incarrying out the methods of the invention, i.e. for producing cells ofthe definitive endoderm from mammalian pluripotent stem cells, such ashuman pluripotent stem cells. A kit according to the invention comprisesat least one DNA demethylating agent.

As noted above, it is understood that the details given herein withrespect to the components employed in the methods of the invention alsoapply to the components comprised by the kits of the invention.

Hence, the at least one DNA demethylating agent comprised by a kit ofthe invention may, for instance, be a cytidine analogue, such as e.g.5-aza-2-deoxycytidine (decitabine), 5-azacytidine (azacitidine),zebularine, Pseudoisocytidine, 5-fluoro-2-deoxycytidine,5,6-dihydro-5-azacytidine, 2′-deoxy-5,6-dihydro-5-azacytidine,6-azacytidine, 2′,2′-Difluoro-deoxycytidine (gemcitabine), orCytosine-beta-D-arabinofurasonide.

The at least one DNA demethylating agent comprised by a kit of theinvention may, for instance, be a cytidine analogue selected from thegroup consisting of 5-aza-2-deoxycytidine (decitabine), 5-azacytidine(azacitidine), 5-fluoro-2-deoxycytidine, 5,6-dihydro-5-azacytidine,2′-deoxy-5,6-dihydro-5-azacytidine, 6-azacytidine and2′,2′-Difluoro-deoxycytidine (gemcitabine),

The at least one DNA demethylating agent comprised by a kit of theinvention may, for instance, be 5-aza-2-deoxycytidine (decitabine) or5-azacytidine (azacitidine), Alternatively, the at least one DNAdemethylating agent comprised by a kit of the invention may, forinstance, be a cytidine analogue which is not 5-aza-2-deoxycytidine(decitabine) or 5-azacytidine (azacitidine).

A kit of the invention may further comprise mammalian pluripotent stemcells, especially human pluripotent stem cells. Hence, a kit of theinvention may comprise mammalian embryonic stem cells, especially humanembryonic stem cells, and/or mammalian induced pluripotent stem cells,especially human induced pluripotent stem cells. The mammalianpluripotent stem cells, especially human pluripotent stem cells, maysuitably be provided as a cell suspension, and may be provided in afrozen state.

A kit of the invention may further comprise activin, such as activin Aor B.

A kit of the invention may further comprise one or more growth factors,such as FGF1, FGF2 and FGF4, and/or serum, such as FBS or FCS.

A kit of the invention may comprise 5-aza-2-deoxycytidine (decitabine)or 5-azacytidine (azacitidine) and mammalian pluripotent stem cells,especially human pluripotent stem cells, such as human embryonic stemcells or human induced pluripotent stem cells.

A kit of the invention may comprise 5-aza-2-deoxycytidine (decitabine)or 5-azacytidine (azacitidine), activin, such activin A or B, andmammalian pluripotent stem cells, especially human pluripotent stemcells, such as human embryonic stem cells or human induced pluripotentstem cells.

The components of a kit of the invention may be provided in the same orseparate containers. For instance, the at least one DNA demethylatingagent and activin may be provided in the same container. If mammalianpluripotent stem cells, especially human pluripotent stem cells, arecomprised by a kit, the mammalian pluripotent stem cells, such as humanpluripotent stem cells are generally provide in a container which isdifferent from the container(s) containing the other components.

The invention also provides compositions. Such compositions areparticularly useful for producing cells of the definitive endoderm frommammalian pluripotent stem cells, especially human pluripotent stemcells. A composition of the invention comprises at least one DNAdemethylating agent and activin, such as activin A or B.

As noted above, it is understood that the details given herein withrespect to the components employed in the methods of the invention alsoapply to the components comprised by the compositions of the invention.

Hence, the at least one DNA demethylating agent comprised by acomposition of the invention may, for instance, be a cytidine analogue,such as e.g. 5-aza-2-deoxycytidine (decitabine), 5-azacytidine(azacitidine), zebularine, Pseudoisocytidine, 5-fluoro-2-deoxycytidine,5,6-dihydro-5-azacytidine, 2′-deoxy-5,6-dihydro-5-azacytidine,6-azacytidine, 2′,2′-Difluoro-deoxycytidine (gemcitabine), orCytosine-beta-D-arabinofurasonide.

The at least one DNA demethylating agent comprised by a composition ofthe invention may, for instance, be a cytidine analogue selected fromthe group consisting of 5-aza-2-deoxycytidine (decitabine),5-azacytidine (azacitidine), 5-fluoro-2-deoxycytidine,5,6-dihydro-5-azacytidine, 2′-deoxy-5,6-dihydro-5-azacytidine,6-azacytidine and 2′,2′-Difluoro-deoxycytidine (gemcitabine),

The at least one DNA demethylating agent comprised by a composition ofthe invention may, for instance, be 5-aza-2-deoxycytidine (decitabine)or 5-azacytidine (azacitidine). Alternatively, the at least one DNAdemethylating agent comprised by a composition of the invention may, forinstance, be a cytidine analogue which is not 5-aza-2-deoxycytidine(decitabine) or 5-azacytidine (azacitidine),

A composition of the invention may further comprise one or more growthfactors, such as FGF1, FGF2 and FGF4, and/or serum, such as FBS or FCS.

Accordingly, a composition of the invention may comprise5-aza-2-deoxycytidine (decitabine) and activin, such as activin A or B.

A composition of the invention may comprise 5-azacytidine (azacitidine)and activin, such as activin A or B.

Definitions

As used herein, “pluripotent” or “pluripotency” refers to the potentialto form all types of specialized cells of the three germ layers(endoderm, mesoderm, and ectoderm); and is to be distinguished from“totipotent” or “totipotency”, that is the ability to form a completeembryo capable of giving rise to offsprings.

As used herein, “human pluripotent stem cells” (hPS) refers to humancells that have the capacity, under appropriate conditions, toself-renew as well as the ability to form any type of specialized cellsof the three germ layers (endoderm, mesoderm, and ectoderm). hPS cellsmay have the ability to form a teratoma in 8-12 week old SCID miceand/or the ability to form identifiable cells of all three germ layersin tissue culture. Included in the definition of human pluripotent stemcells are embryonic cells of various types including human embryonicstem (hES) cells, (see, e.g., Thomson et al. (1998), Heins et.al.(2004), as well as induced pluripotent stem cells [see, e.g. Takahashiet al., (2007); Zhou et al. (2009); Yu and Thomson in Essentials of StemCell Biology (2^(nd) Edition]. The various methods described herein mayutilise hPS cells from a variety of sources. For example, hPS cellssuitable for use may have been obtained from developing embryos by useof a non-destructive technique such as by employing the singleblastomere removal technique described in e.g. Chung et al (2008),further described by Mercader et al. in Essential Stem Cell Methods(First Edition, 2009). Additionally or alternatively, suitable hPS cellsmay be obtained from established cell lines or may be adult stem cells.

As used herein “hiPS cells” refers to human induced pluripotent stemcells. hiPS cells are a type of pluripotent stem cells derived fromnon-pluripotent cells—typically adult somatic cells—by induction of theexpression of genes associated with pluripotency, such as SSEA-3,SSEA-4,TRA-1-60,TRA-1-81,Oct-4, Sox2, Nanog and Lin28.

As used herein, “definitive endoderm (DE)”, “cells of the definitiveendoderm” and “definitive endodermal cells (DE cells)” refers to cellsexhibiting protein and/or gene expression as well as morphology typicalto cells of the definitive endoderm or a composition comprising asignificant number of cells resembling the cells of the definitiveendoderm. The definitive endoderm is the germ cell layer which givesrise to cells of the intestine, pancreas, liver and lung. DE cells maygenerally be characterized, and thus identified, by a positive gene andprotein expression of the endodermal markers FOXA2, CXCR4, HHEX, SOX17,GATA4 and GATA6. The two markers SOX17 and CXCR4 are specific for DE andnot detected in hPS cells. Lastly, DE cells do not exhibit gene andprotein expression of the undifferentiated cell markers Oct4, SSEA-3,SSEA-4, TRA-1-60, TRA-1-81, but can show low Nanog expression.

As used herein, “hepatic progenitors” or “hepatic progenitor cells”refers to cells which have entered the hepatic cell path and give riseto hepatocyte. “Hepatic progenitors” are thus distinguished from“endodermal cells” in that they have lost the potential to develop intocells of the intestine, pancreas and lung. “Hepatic progenitors” maygenerally be characterized, and thus identified, by a positive gene andprotein expression of the early hepatic markers EpCAM, c-Met(HGF-receptor), AFP, CK19, HNF6, C/EBPα and β. They do not exhibit geneand protein expression of the DE-markers CXCR4 and SOX17. Lastly,“hepatic progenitors” do not exhibit gene and protein expression of theundifferentiated cell markers Oct4, SSEA-3, SSEA-4, TRA-1-60 andTRA-1-81 nor the mature hepatic markers CYP1A2, CYP2C9, CYP19, CYP3A4,CYP2B6 and PXR.

As used herein, “hepatocyte” or “hepatocyte-like cells” refers to fullydifferentiated hepatic cells. “Hepatocytes” or “hepatocytes-like cells”may generally be described, and thus identified, by a positive gene andprotein expression of the mature hepatic markers CYP1A2, CYP3A4, CYP2C9,CYP2C19, CYP2B6, GSTA1-1, OATP-2, NTCP, Albumin, PXR, CAR, and HNF4a(isoforms 1+2) among others. Further, “hepatocytes” or “hepatocyte-likecells do not exhibit gene and protein expression of the undifferentiatedcell markers Oct4, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. Compared to DEcells, “hepatocytes” or “hepatocyte-like cells do not exhibit gene andprotein expression of the DE cell markers SOX17 and CXCR4. Compared to“hepatic progenitors”, “hepatocytes” or “hepatocyte-like cells do notexhibit gene and protein expression of the hepatic progenitor markersCytokeratin 19 and AFP.

As meant herein, a gene or protein shall be interpreted as being“expressed”, if in an experiment measuring the expression level of saidgene or protein, the determined expression level is higher than threetimes the standard deviation of the determination, wherein theexpression level and the standard deviation are determined in 10separate determinations of the expression level. The determination ofthe expression level in the 10 separate determinations is preferablycorrected for background-signal.

As used herein HDAC inhibitors refers to Histone deacetylase inhibitors,such as Sodium Butyrate (“NaB”), Phenyl Butyrate (“PB”), Trichostatin Aand Valproic Acid (“VA”).

As used herein, “GSK inhibitor” refers to a compound which inhibits aGlycogen synthase kinase (especially GSK3, including GSK3alpha orGSK3beta).

As used herein, “activator of Wnt signalling” refers to a compound whichactivates Wnt signalling.

As used herein, a DNA demethylating agent is intended to mean a compoundthat interferes with DNA methyltransferase enzyme activity, such asnucleoside analogues, like cytidine analogs, notably5-aza-2-deoxycytidine (decitabine), 5-azacytidine (azacitidine), andzebularine, and non-nucleoside types, such as RG108,S-5-Adenosyl-L-homocysteine, and procaine.

As used herein, the term “FGF” means fibroblast growth factor,preferably of human and/or recombinant origin, and subtypes belongingthereto are e.g. “bFGF” (means basic fibroblast growth factor, sometimesalso referred to as FGF2) and FGF4. “aFGF” means acidic fibroblastgrowth factor (sometimes also referred to as FGF1).

As used herein, the term “Activin” is intended to mean a TGF-beta familymember that exhibits a wide range of biological activities includingregulation of cellular proliferation and differentiation such as“Activin A” or “Activin B”. Activin belongs to the common TGF-betasuperfamily of ligands.

As used herein, the term “ROCK inhibitor” is intended to mean aninhibitor of ROCK Rho-associated protein kinase activity

As used herein the term “xeno-free” is intended to mean completecircumvention of direct or in-direct exposure to non-human animalcomponents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1.

Morphology of hESC-derived definitive endodermal cells (derived withbasic protocol A) without a 5-aza-deoxycytidine treatment during thepre-endodermal phase (day 0-7 of the protocol).

FIG. 1A-2.

Morphology of hESC-derived definitive endodermal cells (derived withbasic protocol A) with a 5-aza-deoxycytidine treatment during thepre-endodermal phase (day 0-7 of the protocol).

FIG. 1B-1.

Morphology of hiPSC-derived definitive endodermal cells (derived withbasic protocol A) without a 5-aza-deoxycytidine treatment during thepre-endodermal phase (day 0-7 of the protocol).

FIG. 1B-2.

Morphology of hiPSC-derived definitive endodermal cells (derived withbasic protocol A) with a 5-aza-deoxycytidine treatment during thepre-endodermal phase (day 0-7 of the protocol).

FIG. 1C-1.

Oct4-immunstaining and DAPI nuclear staining of hiPSC-derived definitiveendodermal cells (derived with basic protocol A) without a5-aza-deoxycytidine treatment during the pre-endodermal phase (day 0-7of the protocol).

FIG. 1C-2.

Oct4-immunstaining and DAPI nuclear staining of hiPSC-derived definitiveendodermal cells (derived with basic protocol D) with a5-aza-deoxycytidine treatment during the pre-endodermal phase (day 0-7of the protocol).

FIG. 1D-1.

mRNA expression of stem cell marker Oct4 in hESC- and hiPSC-deriveddefinitive endodermal cells (derived with basic protocol A) with andwithout a 5-aza-deoxycytidine treatment during the pre-endodermal phase(day 0-7 of the protocol).

FIG. 1D-2.

mRNA expression of stem cell marker Nanog in hESC- and hiPSC-deriveddefinitive endodermal cells (derived with basic protocol A) with andwithout a 5-aza-deoxycytidine treatment during the pre-endodermal phase(day 0-7 of the protocol).

FIG. 1D-3.

mRNA expression of DE marker Sox17 in hESC- and hiPSC-derived definitiveendodermal cells (derived with basic protocol A) with and without a5-aza-deoxycytidine treatment during the pre-endodermal phase (day 0-7of the protocol).

FIG. 1D-4.

mRNA expression of DE marker Cxcr4 in hESC- and hiPSC-derived definitiveendodermal cells (derived with basic protocol A) with and without a5-aza-deoxycytidine treatment during the pre-endodermal phase (day 0-7of the protocol).

FIG. 1D-5.

mRNA expression of DE marker FoxA2 in hESC- and hiPSC-derived definitiveendodermal cells (derived with basic protocol A) with and without a5-aza-deoxycytidine treatment during the pre-endodermal phase (day 0-7of the protocol).

FIG. 1D-6.

mRNA expression of DE marker hHEX in hESC- and hiPSC-derived definitiveendodermal cells (derived with basic protocol A) with and without a5-aza-deoxycytidine treatment during the pre-endodermal phase (day 0-7of the protocol).

FIG. 1D-7.

mRNA expression of extraembryonic marker Sox7 in hESC- and hiPSC-deriveddefinitive endodermal cells (derived with basic protocol A) with andwithout a 5-aza-deoxycytidine treatment during the pre-endodermal phase(day 0-7 of the protocol).

FIG. 2A.

mRNA expression of stem cell marker Oct4 in definitive endodermal cellsderived from 27 different hESC- and hiPSC lines (derived with basicprotocols A and B, respectively) with a 5-aza-deoxycytidine treatmentduring the pre-endodermal phase (day 0-7 of the protocol).

FIG. 2B.

mRNA expression of stem cell marker Nanog in definitive endodermal cellsderived from 27 different hESC- and hiPSC lines (derived with basicprotocols A and B, respectively) with a 5-aza-deoxycytidine treatmentduring the pre-endodermal phase (day 0-7 of the protocol).

FIG. 2C.

mRNA expression of DE marker Sox17 in definitive endodermal cellsderived from 27 different hESC- and hiPSC lines (derived with basicprotocols A and B, respectively) with a 5-aza-deoxycytidine treatmentduring the pre-endodermal phase (day 0-7 of the protocol).

FIG. 2D.

mRNA expression of DE marker Cxcr4 in definitive endodermal cellsderived from 27 different hESC- and hiPSC lines (derived with basicprotocols A and B, respectively) with a 5-aza-deoxycytidine treatmentduring the pre-endodermal phase (day 0-7 of the protocol).

FIG. 3A.

mRNA expression of stem cell marker Oct4 in definitive endodermal cellsderived from 3 hESC- and hiPSC lines (derived with basic protocols A andB, respectively) with or without a treatment with 5-aza-deoxycytidine or5-azacytidine during the pre-endodermal phase (day 0-7 of the protocol.

FIG. 3B.

mRNA expression of stem cell marker Nanog in definitive endodermal cellsderived from 3 hESC- and hiPSC lines (derived with basic protocols A andB, respectively) with or without a treatment with 5-aza-deoxycytidine or5-azacytidine during the pre-endodermal phase (day 0-7 of the protocol).

FIG. 3C.

mRNA expression of DE marker Sox17 in definitive endodermal cellsderived from 3 hESC- and hiPSC lines (derived with basic protocols A andB, respectively) with or without a treatment with 5-aza-deoxycytidine or5-azacytidine during the pre-endodermal phase (day 0-7 of the protocol).

FIG. 3D.

mRNA expression of DE marker Cxcr4 in definitive endodermal cellsderived from 3 hESC- and hiPSC lines (derived with basic protocols A andB, respectively) with or without a treatment with 5-aza-deoxycytidine or5-azacytidine during the pre-endodermal phase (day 0-7 of the protocol).

EXAMPLES

Examples of general culturing and passaging techniques are disclosed inapplications WO2004/099394, WO2003/055992, WO2007/042225, WO2007/140968and WO2011116930.

As laid out in the following examples, the starting material are humanpluripotent stem cells, in particular human embryonic stem cells andhuman induced pluripotent stem cells.

Example 1 Maintenance of hPS Cell Types

All hPS cells (as defined above) can be used as staring material forthis invention. For the examples below in particular definitive endodermwas derived in vitro from undifferentiated human embryonic stem cells(hESC) established on mEF feeder cells (Heins et al 2004) and maintainedunder feeder-free conditions. The cell lines used for this experimentcould be, but are not limited to the hES cell lines SA167, SA181, SA461(Cellartis AB, Göteborg, Sweden) and they can be propagated as describedby Heins et al. 2004. These cell lines are listed in the NIH stem cellregistry, the UK Stem Cell bank and the European hESC registry and areavailable on request.

Along with hPS obtained from hESC, hiPS (human induced pluripotent stem)cells have also been used for the derivation of hepatocytes for theexamples of this invention.

The hiPSC line used in this invention are derived as followed: Humandermal fibroblasts (CRL2429, ATCC) were maintained in DMEM supplementedwith 10% fetal bovine serum, 1× glutamax, 5 U/ml penicillin and 5 μg/mlstreptomycin at 37° C. in a humidified atmosphere of 5% CO₂ in air.Fibroblasts were tranduced with recombinant lentiviruses encoding mouseOct4, Sox2, Klf4 and c-myc and cultured for 5 days. The transduced cellswere then dispersed with trypsin and seeded onto mitomycin C treatedhuman dermal fibroblast feeder cells at a density of 5×10³ cells/cm² intheir normal growth medium. After 24 hours the medium was replaced withknockout DMEM supplemented with 20% knockout serum replacement, 1×non-essential amino acids, 1× glutamax, 5 U/ml penicillin, 5 μg/mlstreptomycin, 100 μM 2-mercaptoethanol and 30 ng/ml bFGF at 37° C. in ahumidified atmosphere of 5% CO₂ in air. Half of the volume of medium wasreplaced every day and colonies of iPS cells emerged after approximately30 days. iPS colonies were picked, expanded in DEF-CS™, and cell banksprepared. The banked cells were then characterised to check for theexpression of endogenous Oct4, Sox2, Klf4 and c-Myc, silencing oftransgenes, potential to differentiate into cell types representative ofall three germ layers in vitro, and to confirm their authenticity by STRprofiling and comparison with the parental fibroblast cell line (ATCC).Alternatively to reprogramming using lentivirus, hiPSC lines can also bereprogrammed using retrovirus, Sendai virus, adenovirus, episomalplasmid vectors, proteins and mRNAs or other techniques. Other suitablecell lines for use are those established by Chung et al. (2008), such ascell lines MA126, MA127, MA128 and MA129 (Advanced Cell Technology, Inc.Worcester, Mass., USA), which all are listed with the International stemcell registry. These cell lines have been derived (or obtained) withoutdestruction of the human embryo by employing a single blastomere removaltechnique.

Example 2 Differentiation of hPS Cell Types to Produce Hepatocyte-LikeCells

Hepatocyte-like cells may be derived from hPS cells by employing thefollowing exemplary basic protocols A and B:

Protocol A:

Undifferentiated hPS cells are dissociated and seeded directly infreshly prepared day 0-medium. The different mediums were preparedfreshly and added day 0, 1, 2, 3, 4, 5, 7. The pre-treatment medium isavailable from Cellectis AB (Arvid Wallgrens Backe 20, 41356 Gothenburg,Sweden).

Day 0

-   Pre-treatment medium-   3 μM CHIR99021-   5 μM ROCK inhibitor

Day 1

-   Pre-treatment medium-   3 μM CHIR99021

Day 2

-   RPMI 1640 (+0.1% PEST+1% Glutamax)-   1×B27-   50 ng/ml Activin A-   3 μM CHIR99021-   5 μM LY294002

Day 3

-   RPMI 1640 (+0.1% PEST+1% Glutamax)-   1×B27-   50 ng/ml Activin A-   5 μM LY294002

Day 4-7

-   RPMI 1640 (+0.1% PEST+1% Glutamax)-   1×B27-   50 ng/ml Activin A    On day 7 the cells are passaged. The cells are incubated for 3-7    minutes with TrypLE Select at 37° C., the same volume of VitroHES is    added and the cell suspension is centrifuged at 200-300 g, 5-6 min.    Thereafter, the cells are replated onto a Fibronectin-based coating    at a cell density of 50 000-350 000 cells/cm² such as e.g. 100    000-300 000 cells/cm², preferably 150 000 cells/cm².

Protocol B:

Undifferentiated hPS cells are dissociated and seeded directly infreshly prepared day 0-medium. The different mediums were preparedfreshly and added day 0, 1, 2, 3, 4, 5, 7. The pre-treatment medium isavailable from Cellectis AB (Arvid Wallgrens Backe 20, 41356 Gothenburg,Sweden).

-   Day 0-   Pre-treatment medium-   3 μM CHIR99021-   5μM ROCK inhibitor-   Day 1-   RPMI 1640 (+0.1% PEST+1% Glutamax)-   1×B27-   50 ng/ml Activin A-   3 μM CHIR99021-   5 μM LY294002-   Day 2-   RPMI 1640 (+0.1% PEST+1% Glutamax)-   1×B27-   50 ng/ml Activin A-   5 μM LY294002-   Day 3-7-   RPMI 1640 (+0.1% PEST+1% Glutamax)-   1×B27-   50 ng/ml Activin A

For passage d7 see Protocol A.

Example 3 Validation of Improved Definitive Endoderm Phenotype in hESCand hiPS Cells Treated with DNA Demethylation Procedure:

Following the basic protocol A (both for hESC- and hiPSC-deriveddefinitive endoderm), cells were treated with 10 nM5-aza-2-deoxycytidine at different time points and for differentdurations during the pre-endodermal phase, e.g. on day 2-3, 2-4, 3-4 and4-6 of the protocol (hESC-DE: no 5azadC n=4, 5azadC d2-3 n=4, d3-4 n=1,d2-4 n=1, d4-6 n=1; hiPSC-DE: no 5azadC n=5, 5azadC d2-3 n=5, d3-4 n=2,d2-4 n=1, d4-6 n=1; with n being the number of individual experiments).

For analysis of mRNA expression, hESC- and hiPSC-derived DE-cells wereharvested on day 7 of the protocol and gene expression was analysedusing qRT-PCR, normalised to the house-keeping gene CREBBP, and theresults presented as relative quantification normalised to a calibrator.

Results: FIG. 1A:

DE derived from hESC treated with 10 nM 5-aza-2-deoxycytidine on day 2-3(FIG. 1 A2) is more homogeneous and has more pronounced cell-cellcontacts compared to untreated control DE (FIG. 1 A1). Note the presenceof undifferentiated cells in the control DE (FIG. 1 A1) which is inaccordance with higher expression of Oct4 and Nanog mRNA expression incontrol DE (compare FIG. 1D). Similar results were obtained whentreating cells on days 2-4, 3-4 and 4-6 and with 100 nM5-aza-2-deoxycytidine. 1 nM 5-aza-2-deoxycytidine had less effect (datanot shown).

FIG. 1B:

HiPSC-derived DE treated with 10 nM 5-aza-2-deoxycytidine on day 2-3(FIG. 1 B2) is more confluent and has more pronounced cell-cell contactsthan control DE (FIG. 1 B1). Similar results were obtained when treatingcells days 2-4, 3-4 and 4-6 and with 100 nM 5-aza-2-deoxycytidine. 1 nM5-aza-2-deoxycytidine had less effect (data not shown).

FIG. 1C:

HiPSC-derived DE treated with 10 nM 5-aza-2-deoxycytidine on day 2-3 hasmuch less Oct4-immunopositive cells at day 7 compared to untreatedcontrols, i.e. less undifferentiated cells are left and the DE is morehomogeneous after treatment with a demethylating agent.

FIG. 1D:

Expression of the stem cell marker Oct4 is much lower in hESC- andhiPSC-derived DE treated with 10 nM 5azadC on day 2-3, 3-4, 2-4, and 4-6than in untreated controls (FIG. 1 D1). In 5azadC-treated hESC-derivedDE mRNA expression of the stem cell marker Nanog is strongly decreasedwhereas it remains mainly unaffected in hiPSC-derived DE (FIG. 1 D1).Expression of the DE markers Sox17, Cxcr4, FoxA2 and hHex isup-regulated in 5azadC-treated hESC- and hiPSC-derived DE compared tountreated controls while the effect is stronger in hESC-derived DE thanin hiPSC-derived DE (FIG. 1 D3-6). Expression of the extraembryonicmarker Sox7 is very low both in control and 5azadC-treated hESC- andhiPSC-derived DE with the exception of 5azadC-treatment on days 2-4 and4-6 which increases Sox7 mRNA levels.

Taken together, the treatment of the cells with a DNA demethylationagent during endodermal development led to improved DE morphology and DEcell yield in both hESC and hiPSC derived cells (FIG. 1 A-B).Furthermore it resulted in a stronger decrease of the stem cell markerOct4 as detected by immunocytochemistry (FIG. 1 C), to an improvedexpression of well defined DE markers SOX17, CXCR4, HEX, Foxa2, as wellas a decrease of the extraembryonic endoderm marker Sox7 and of the stemmarkers Oct4 and Nanog (FIG. 1 D). Therefore the skilled person wishingto produce a more homogeneous population of definitive endoderm cellscan select from one or more DNA-demethylation agents and employ theme.g. at days 2-3 or 3-4 during differentiation of pluripotent stem celltypes.

Example 4 Highly Homogeneous Definitive Endoderm Derived from a Panel of27 hPSC Lines by Treatment with DNA Demethylating Agents during DEDifferentiation Procedure:

Following the basic protocols A or B, cells derived from 27 hPSC lineswere treated with 10 nM 5-aza-2-deoxycytidine on day 2-3 during duringthe hPS differentiation into definitive endoderm (protocol A: ChiPSC14,ChiPSC19, ChiPSC22, P11015, SA167, SA181, SA461, and Val9; protocol B:ChiPSC4, ChiPSC6b, ChiPSC7, ChiPSC8, ChiPSC9, ChiPSC10, ChiPSC11,ChiPSC13, ChiPSC15, ChiPSC17, ChiPSC18, ChiPSC19, ChiPSC20, ChiPSC21,ChiPSC23, ChiPSC24, P11012, P11021, P11025, and SA121).

23 out of 27 hPSC lines were tested with both protocols A and B. Out ofthese 2-3 lines, only 4 cell lines (ChiPSC14, ChiPSC23, P11015, andP11032) could only be differentiated with one of the two protocols. FourhPSC lines (ChiPSC8, ChiPSC9, ChiPSC10, and ChiPSC11) were only testedwith protocol B.

For analysis of mRNA expression, hESC- and hiPSC-derived DE-cells wereharvested on day 7 of the protocol and gene expression was analysedusing qRT-PCR, normalised to the house-keeping gene CREBBP, and theresults presented as relative quantification normalised to a calibrator.

Results: FIG. 2A-D:

Using the basic protocols A or B including a DNA demethylating treatmenton day 2-3 during the hPS differentiation into definitive endoderm,undifferentiated stem cells from 27 different hPSC lines could bedifferentiated into highly homogeneous DE displaying low mRNA expressionlevels of the stem cell markers Oct-4 and Nanog (FIG. 2A, B) and highlevels of the DE markers Sox17 and Cxcr4 (FIG. 2C, D) compared toundifferentiated hESC (SA181) and hiPSC (ChiPSC4).

Taken together, the treatment of the cells during hPS differentiationinto definitive endoderm with a DNA demethylating agent allowsderivation of homogeneous DE with low expression levels of stem cellmarkers and high expression levels of DE markers from all hPSC linestested. The derivation of homogeneous DE is crucial for derivation ofhomogeneous hepatocyte cultures which could be obtained from all linestested (data not shown).

Therefore the skilled person wishing to produce a homogeneous populationof definitive endoderm cells from any given hPSC line can include atreatment with a DNA demethylating agent, for instance, on day 2-3during differentiation of hPS cells into definitive endodem.

Example 5 Both DNA Demethylating Agents 5-aza-2-deoxycytidine and5-azacytidine Improve the Definitive Endoderm Phenotype in hESC- andhiPSC-Derived DE Procedure:

Following the basic protocols A (hPSC lines P11032, and SA181) or B(hPSC line P11012), cells derived from 3 different hPSC lines weretreated with either 10 nM 5-aza-2-deoxycytidine or 1 μM 5-azacytidine onday 2-3 during the hPS differentiation into definitive endodem.

For analysis of mRNA expression, hESC- and hiPSC-derived DE-cells wereharvested on day 7 of the protocol and gene expression was analysedusing qRT-PCR, normalised to the house-keeping gene CREBBP, and theresults presented as relative quantification normalised to a calibrator.

Results: FIG. 3:

A, B) Without treatment with a demethylating agent, the three hPSC linesP11032, SA181 and P11012 produced heterogeneous DE with relatively highmRNA expression of stem cell markers Oct4 and Nanog (FIG. 3A, B).Treatment with the DNA demethylating agents 5-aza-2-deoxycytidine(5azadC) and 5-azacytidine (5azaC) significantly decreased Oct4 andNanog mRNA (FIG. 3 A, B) and thus allowed derivation of a homogeneous DEpopulation from these three hPSC lines.

C, D) No significant changes in mRNA expression of the DE markers Sox17and Cxcr4 could be observed upon treatment with 10 nM5-aza-2-deoxycytidine or 1 μM 5-azacytidine (FIG. 3 C, D).

Taken together, treatment with both DNA demethylating agents5-aza-2-deoxycytidine (5azadC) and 5-azacytidine (5azaC) allowsderivation of homogeneous DE from hPSC lines, giving otherwiseheterogeneous DE if untreated.

Therefore the skilled person wishing to produce a homogeneous populationof definitive endoderm cells can select from one or moreDNA-demethylation agents and employ them e.g. at days 2-3 duringdifferentiation of pluripotent stem cell types into definitive endodem.

REFERENCES

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1. A composition comprising at least one DNA demethylating agent andactivin.
 2. The composition according to claim 1, wherein the DNAdemethylating agent is a cytidine analogue.
 3. The composition accordingto claim 1, wherein the DNA demethylating agent is selected from thegroup consisting of 5-aza-2-deoxycytidine (decitabine), 5-azacytidine(azacitidine), zebularine, Pseudoisocytidine, 5-fluoro-2-deoxycytidine,5,6-dihydro-5-azacytidine, 2′-deoxy-5,6-dihydro-5-azacytidine,6-azacytidine, 2′,2′-Difluoro-deoxycytidine (gemcitabine), orCytosine-beta-D-arabinofurasonide.
 4. The composition according to claim1, wherein the DNA demethylating agent is selected from the groupconsisting of 5-aza-2-deoxycytidine (decitabine), 5-azacytidine(azacitidine), 5-fluoro-2-deoxycytidine, 5,6-dihydro-5-azacytidine,2′-deoxy-5,6-dihydro-5-azacytidine, 6-azacytidine and2′,2′-Difluoro-deoxycytidine (gemcitabine).
 5. The composition accordingto claim 1, wherein the DNA demethylating agent is 5-aza-2-deoxycytidine(decitabine).
 6. The composition according to claim 1, wherein the DNAdemethylating agent is 5-azacytidine (azacitidine).
 7. The compositionaccording to claim 1, wherein the activin is activin A or activin B. 8.The composition according to claim 1, further comprising one or moregrowth factors and/or serum.
 9. The composition according to claim 8,wherein the one or more growth factors is selected from the groupconsisting of FGF1, FGF2 and FGF4.
 10. A kit comprising at least one DNAdemethylating agent and activin.
 11. The kit according to claim 10,wherein the DNA demethylating agent is a cytidine analogue.
 12. The kitaccording to claim 10, wherein the DNA demethylating agent is selectedfrom the group consisting of 5-aza-2-deoxycytidine (decitabine),5-azacytidine (azacitidine), zebularine, Pseudoisocytidine,5-fluoro-2-deoxycytidine, 5,6-dihydro-5-azacytidine,2′-deoxy-5,6-dihydro-5-azacytidine, 6-azacytidine,2′,2′-Difluoro-deoxycytidine (gemcitabine), orCytosine-beta-D-arabinofurasonide.
 13. The kit according to claim 10,wherein the DNA demethylating agent is selected from the groupconsisting of 5-aza-2-deoxycytidine (decitabine), 5-azacytidine(azacitidine), 5-fluoro-2-deoxycytidine, 5,6-dihydro-5-azacytidine,2′-deoxy-5,6-dihydro-5-azacytidine, 6-azacytidine and2′,2′-Difluoro-deoxycytidine (gemcitabine).
 14. The kit according toclaim 10, wherein the DNA demethylating agent is 5-aza-2-deoxycytidine(decitabine).
 15. The kit according to claim 10, wherein the DNAdemethylating agent is 5-azacytidine (azacitidine).
 16. The kitaccording to claim 10, wherein the activin is activin A or B.
 17. Thekit according to claim 10, further comprising one or more growth factorsand/or serum.
 18. The kit according to claim 17, wherein the one or moregrowth factors is selected from the group consisting of FGF1, FGF2 andFGF4.
 19. The kit according to claim 10, further comprising mammalianpluripotent stem cells.
 20. The kit according to claim 19, wherein themammalian pluripotent stem cells are human pluripotent stem cells.